PROJECT SUMMARY Red blood cells are the most abundant cell type in the human body, accounting for 25 out of 30 trillion total human cells in an adult individual. During their 120-day life span in the circulatory system, red blood cells play a vital role in the transport of oxygen and its delivery to tissues, a function that is finely tuned by red blood cell metabolism. By leveraging novel technology generated in the lab (?high-throughput metabolomics?) and an international network of ~45 collaborators (providing a total of ~20,000 samples from research and clinical cohort studies), we will investigate how factors such as aging, genetics, environment and storage impact red blood cell metabolism and, in so doing, influence tissue oxygenation and systems physiology in health and disease. At completion of this hypothesis-generating project, we will have defined how and to what extent red blood cell metabolism is modulated as a function of: (i) Aging, from intra-uterine to elderly life, from erythropoiesis to senescent red blood cells; (ii) Genetics, including sex (chromosome X/Y), ethnicity, species (by investigating red cells from zoo animals and research animal models) ? aneuploidy (Down syndrome) or mutations that impact the activity of enzymes involved in red cell metabolism (including glucose 6-phosphate dehydrogenase deficiency ? the most common enzymopathy in humans that impacts ~400 million people) and oxygen transport, such as Sickle Cell Disease, beta-thalassemia; (iii) Environment, including diet, iron availability, exercise, hormonal therapy in subjects undergoing sex exchange therapy, microbiome, infections (either bacterial or viral infections ? including malaria, Zika, Dengue, Chikungunya virus), conditions related to hypoxia (such as high-altitude, respiratory diseases, ischemic or hemorrhagic shock, cancer, radio and chemotherapy) and drugs; (iv) Storage, in the largest available clinical cohort (13,800 donors) and animal models. The project will pave the way for the generation of RBC Atlas, an online repository for the dissemination of metabolic data from red blood cell-related studies, a critical step towards Personalized Transfusion Medicine. Finally, we will investigate mechanisms of metabolic regulation mediated by the ?AE1-Hb switch?, a phenomenon that involves the most abundant red blood cell proteins in the cytosol and membrane, hemoglobin and band 3 (AE1), respectively. Knowledge and technology developed in this study will inform our understanding of red cell metabolic regulation in health and disease, as well as our capacity to manipulate it for diagnostic and therapeutic purposes. Translation of expected findings will impact diverse endeavors, from clinical biochemistry to personalized transfusion medicine, from hematology to veterinary medicine, from pulmonology to virology, from sports physiology to cancer medicine.